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Assessing long-distance RNA sequence connectivity via RNA-templated DNA-DNA ligation.

Roy CK, Olson S, Graveley BR, Zamore PD, Moore MJ - Elife (2015)

Bottom Line: Multiple sites of alternative splicing within a single gene exponentially increase the number of possible spliced isoforms, with most human genes currently estimated to express at least ten.To understand the mechanisms underlying these complex isoform expression patterns, methods are needed that faithfully maintain long-range exon connectivity information in individual RNA molecules.Using this assay, we test proposed coordination between distant sites of alternative exon utilization in mouse Fn1, and we characterize the extraordinary exon diversity of Drosophila melanogaster Dscam1.

View Article: PubMed Central - PubMed

Affiliation: RNA Therapeutics Institute, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, United States.

ABSTRACT
Many RNAs, including pre-mRNAs and long non-coding RNAs, can be thousands of nucleotides long and undergo complex post-transcriptional processing. Multiple sites of alternative splicing within a single gene exponentially increase the number of possible spliced isoforms, with most human genes currently estimated to express at least ten. To understand the mechanisms underlying these complex isoform expression patterns, methods are needed that faithfully maintain long-range exon connectivity information in individual RNA molecules. In this study, we describe SeqZip, a methodology that uses RNA-templated DNA-DNA ligation to retain and compress connectivity between distant sequences within single RNA molecules. Using this assay, we test proposed coordination between distant sites of alternative exon utilization in mouse Fn1, and we characterize the extraordinary exon diversity of Drosophila melanogaster Dscam1.

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Comparison of RT-PCR and ligation-based Dscam1 isoform analysis techniques.(A) Individual exon usage in S2 cells as measured by SeqZip and CAMSeq. Gray rectangles indicate exon variants (6.47 and 9.31) whose apparent inclusion frequency was substantially different between SeqZip and CAMSeq. (B) Scatter plot of isoform expression measured by SeqZip and CAMSeq. Gray areas indicate the off-axis populations containing exon variants 6.47 and 9.31.DOI:http://dx.doi.org/10.7554/eLife.03700.016
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fig7s1: Comparison of RT-PCR and ligation-based Dscam1 isoform analysis techniques.(A) Individual exon usage in S2 cells as measured by SeqZip and CAMSeq. Gray rectangles indicate exon variants (6.47 and 9.31) whose apparent inclusion frequency was substantially different between SeqZip and CAMSeq. (B) Scatter plot of isoform expression measured by SeqZip and CAMSeq. Gray areas indicate the off-axis populations containing exon variants 6.47 and 9.31.DOI:http://dx.doi.org/10.7554/eLife.03700.016

Mentions: We next used SeqZip to measure Dscam1 isoform identity and abundance in S2 cells, as well as 4–6 hr and 14–16 hr D. melanogaster embryos. Ligamers targeting every exon variant in clusters 4, 6, and 9 together with ligamers for constitutive exons 3, 5, 7, 8, and 10 (97 ligamers in all) reduced the median size of mRNA sequences analyzed from 1734 nt (1722–1751 nt for exons 3–10) to 356 nt for a seven-ligamer product formed by six ligation events. This approximately fivefold length reduction allowed the products to be fully sequenced using 250 bp, paired-end reads in a single Illumina MiSeq run (Figure 4C). Between 449,113 and 946,110, high-confidence alignments were obtained for each sample (Supplementary file 2). Across all three samples, SeqZip detected 8397 of the 18,612 possible isoforms (Figure 6A). Individual isoform abundances were highly correlated between both technical and biological replicates (r = 0.8–0.95, p < 2.2 × 10−16, Fisher z-transformation; Figure 6—figure supplement 1). Of the 97 possible exons represented in our ligamer set, all were detected except exon 6.11, which is generally thought to be an unused pseudo-exon (Neves et al., 2004; Zhan et al., 2004; Watson et al., 2005; Miura et al., 2013; Sun et al., 2013). The absence of exon 6.11 reads from our libraries provides additional evidence for the specificity of SeqZip. Further, with two exceptions, the patterns of individual exon use in S2 cells were directly comparable between the SeqZip and CAMSeq data sets (r = 0.87, p < 2.2 × 10−16, Fisher z-transformation; Figure 7—figure supplement 1): exon 6.47 was well represented in the CAMSeq data but undetectable by SeqZip, and exon 9.31 was more abundantly represented in our data.10.7554/eLife.03700.013Figure 6.SeqZip captures diverse Dscam1 isoform expression and exon use.


Assessing long-distance RNA sequence connectivity via RNA-templated DNA-DNA ligation.

Roy CK, Olson S, Graveley BR, Zamore PD, Moore MJ - Elife (2015)

Comparison of RT-PCR and ligation-based Dscam1 isoform analysis techniques.(A) Individual exon usage in S2 cells as measured by SeqZip and CAMSeq. Gray rectangles indicate exon variants (6.47 and 9.31) whose apparent inclusion frequency was substantially different between SeqZip and CAMSeq. (B) Scatter plot of isoform expression measured by SeqZip and CAMSeq. Gray areas indicate the off-axis populations containing exon variants 6.47 and 9.31.DOI:http://dx.doi.org/10.7554/eLife.03700.016
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4442144&req=5

fig7s1: Comparison of RT-PCR and ligation-based Dscam1 isoform analysis techniques.(A) Individual exon usage in S2 cells as measured by SeqZip and CAMSeq. Gray rectangles indicate exon variants (6.47 and 9.31) whose apparent inclusion frequency was substantially different between SeqZip and CAMSeq. (B) Scatter plot of isoform expression measured by SeqZip and CAMSeq. Gray areas indicate the off-axis populations containing exon variants 6.47 and 9.31.DOI:http://dx.doi.org/10.7554/eLife.03700.016
Mentions: We next used SeqZip to measure Dscam1 isoform identity and abundance in S2 cells, as well as 4–6 hr and 14–16 hr D. melanogaster embryos. Ligamers targeting every exon variant in clusters 4, 6, and 9 together with ligamers for constitutive exons 3, 5, 7, 8, and 10 (97 ligamers in all) reduced the median size of mRNA sequences analyzed from 1734 nt (1722–1751 nt for exons 3–10) to 356 nt for a seven-ligamer product formed by six ligation events. This approximately fivefold length reduction allowed the products to be fully sequenced using 250 bp, paired-end reads in a single Illumina MiSeq run (Figure 4C). Between 449,113 and 946,110, high-confidence alignments were obtained for each sample (Supplementary file 2). Across all three samples, SeqZip detected 8397 of the 18,612 possible isoforms (Figure 6A). Individual isoform abundances were highly correlated between both technical and biological replicates (r = 0.8–0.95, p < 2.2 × 10−16, Fisher z-transformation; Figure 6—figure supplement 1). Of the 97 possible exons represented in our ligamer set, all were detected except exon 6.11, which is generally thought to be an unused pseudo-exon (Neves et al., 2004; Zhan et al., 2004; Watson et al., 2005; Miura et al., 2013; Sun et al., 2013). The absence of exon 6.11 reads from our libraries provides additional evidence for the specificity of SeqZip. Further, with two exceptions, the patterns of individual exon use in S2 cells were directly comparable between the SeqZip and CAMSeq data sets (r = 0.87, p < 2.2 × 10−16, Fisher z-transformation; Figure 7—figure supplement 1): exon 6.47 was well represented in the CAMSeq data but undetectable by SeqZip, and exon 9.31 was more abundantly represented in our data.10.7554/eLife.03700.013Figure 6.SeqZip captures diverse Dscam1 isoform expression and exon use.

Bottom Line: Multiple sites of alternative splicing within a single gene exponentially increase the number of possible spliced isoforms, with most human genes currently estimated to express at least ten.To understand the mechanisms underlying these complex isoform expression patterns, methods are needed that faithfully maintain long-range exon connectivity information in individual RNA molecules.Using this assay, we test proposed coordination between distant sites of alternative exon utilization in mouse Fn1, and we characterize the extraordinary exon diversity of Drosophila melanogaster Dscam1.

View Article: PubMed Central - PubMed

Affiliation: RNA Therapeutics Institute, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, United States.

ABSTRACT
Many RNAs, including pre-mRNAs and long non-coding RNAs, can be thousands of nucleotides long and undergo complex post-transcriptional processing. Multiple sites of alternative splicing within a single gene exponentially increase the number of possible spliced isoforms, with most human genes currently estimated to express at least ten. To understand the mechanisms underlying these complex isoform expression patterns, methods are needed that faithfully maintain long-range exon connectivity information in individual RNA molecules. In this study, we describe SeqZip, a methodology that uses RNA-templated DNA-DNA ligation to retain and compress connectivity between distant sequences within single RNA molecules. Using this assay, we test proposed coordination between distant sites of alternative exon utilization in mouse Fn1, and we characterize the extraordinary exon diversity of Drosophila melanogaster Dscam1.

Show MeSH